Lysozymes are a unique class of enzymes which can be extracted from a variety of organic sources, but most profitably from hen egg-whites (HEW). Physical and biochemical aspects of HEW lysozyme have been extensively researched since lysozyme was first discovered by Sir Alexander Fleming in 1922. This analysis has shown that lysozyme is a small but hardy enzyme which has a potent antibacterial action (which accounts for its current pharmaceutical importance).
Work involving use of lysozyme in foods has been conducted mainly in Japan. Lysozyme can be used on food packaging films to render the films antiseptic, and can be added to foods, such as sausages, other meats, and dried milk compositions, to serve as a preservative.
Purified lysozyme or its salts are of decided commercial value in Japanese and world markets. Certain investigators have claimed that lysozymes are beneficial in the treatment of cancers. Continuing research and interest in lysozymes encourages the development of a commercial method to isolate quantities of the proteins.
Egg-white consists of a mixture of nearly pure protein having ovalbumin, conalbumin, ovomucoid, lysozyme, ovomucin, flavoprotein-apoprotein, "proteinase inhibitor," avidin, other proteins, and nonproteins. The other proteins mainly include globulins, while the nonproteins are primarily glucose and poorly characterized salts. For purposes of this description, egg-white may be characterized as being a lysozyme fraction (consisting essentially of lysozyme) and an albumin fraction (consisting of the other proteins and nonproteins). In this description, "albumin" is used to describe the bulk of the egg white and is intended to be synonomous with "albumen," which is also commonly used as a noun for the egg white. The "albumin fraction" is the egg white remaining after extraction of the lysozyme.
Lysozyme is an enzyme, and causes cell lysis (dissolution) by breaking bonds in carbohydrate polymers found in the cell walls. Lysozyme has antibiotic activity. In egg-white, lysozyme is the active ingredient to protect the embryo against microbial factors.
HEW lysozyme is a basic protein of pI 10.5, molecular weight approximately 14,000 Daltons, and S.sub.20, W value of 1.9. H. FEVOLD, ADVANCES IN PROTEIN CHEMISTRY, 6, 188-252 (1951). The lysozyme apparently cleaves the beta-glucosidic linkage between the C1 of N-acetylmuramic acid and the C4 of N-acetylglucosamine polymers.
Lysozymes are used clinically as antibacterialitic agents against infectious diseases, therapeutic agents in the treatment of wounds, and potentiators of many antibiotics. That is, lysozyme increases the effect of the antibiotics and allows lower concentrations of antibiotics to act for longer periods of time.
Because dried egg-whites are commercially valuable apart from the lysozyme, it is important that a commercial method be developed to allow the extraction of the lysozyme while retaining the commercial value of the remaining egg-white. Furthermore, a desirable method would include use of a reusable resin of the affinity type to allow recovery of the lysozyme at a reduced cost. Because lysozyme is capable of cleaving the NAM-NAG bond of murein to cause cell lysis, immobilizing lysozyme on an affinity resin often leads to hydrolysis of the resin. A resin to which lysozyme could be reversibly bound would provide a foundation for a commercially valuable process for the separation of lysozyme from egg-white.
U.S. Pat. No. 3,515,643 discloses a process for the production of lysozyme which consists of contacting the egg-white with a weakly acidic ion-exchange resin at a pH of 6-7 to preferentially bind the lysozyme by ionic action. Preferred exchange resins are "Amberlite CG-50" and "Amberlite IRC-50." (Both are methacrylic carboxylic acid resins obtainable from Rohm and Haas Company. "Amberlite" is a registered trademark.) Ion-exchange resins differ markedly in their mechanism of action from that of affinity resins. Ion-exchange resins work on the basis of electrostatic charge, while affinity resins work on the basis of chemical structure.
U.S. Pat. No. 3,419,471 discloses a method for preparation of albumin-free lysozyme using an ion-exchange filtration method. Anion exchange cellulose or anion exchange dextran previously buffered with a buffer solution of the same pH and ionic strength as the enzyme solution to be treated are used in one step of the process. Suitable anion exchange celluloses include GE-cellulose, TEAE-cellulose, DEAE-cellulose, and AE-cellulose. DEAE-Sephadex A-50 is a suitable anion exchange dextran. The pH is maintained between about 6.0-10.5, preferably between about 6.5-10.0, to allow preferential adsorption of albumins to the resin, leaving a substantially albumin-free lysozyme extract. The lysozyme must be desalted prior to ion exchange. A double-bed ion exchange for desalting and extraction uses a cation exchange resin followed by an anion exchange resin. This method maintains lysozyme activity even at low pH, where activity would otherwise be irreversibly reduced.
Muzzarelli discloses the preferential, reversible adsorption of lysozyme on chitosan. The chitosan is not hydrolyzed by the lysozyme. Muzzarelli suggests eluting the chitosan with a propylamine solution and suggests pretreating HEW with sulfuric acid prior to adsorption on the chitosan to eliminate high molecular weight egg-white proteins. The albumin fraction is irreversibly damaged with this process.
Jensen and Kleppe disclose an affinity between lysozyme and chitin, but show that lysozyme destroys the chitin resin by hydrolyzing the N-acetyl-D-glycosamine residues of the chitin.
Cherkasov discloses a method of separating lysozyme from HEW by diluting the HEW, adjusting the pH to 5.5, and contacting the treated solution with chitin powder at a pH between 5.0 and 5.5 and an ion strength greater than or equal to 0.1. Cherkasov teaches that at pH 7.9-8.0, lysozyme complexes with chitin to strongly bind to the chitin resin and the affinity can only be broken with dilute acid, such as 0.1M acidic acid.
Weaver discloses that deaminated chitin has a high specificity and capacity for lysozyme. Deaminated chitin has a good stability for isolating lysozyme and allows fast flow rates.
Tosa discloses that kappa-carrageenan is a polysaccharide composed of unit structures of beta-D-galactose sulfate and 3,6-anhydro-alpha-D-galactose, having a molecular weight of around 100,000-800,000 Daltons and an ester content of 20-30%. The article details that kappa-carrageenan is a suitable polysaccharide for immobilization of enzymes and microbial cells in its gel lattice. As described by Chibata, kappa-carrageenan shows a unique ability to bind with and to stabilize proteins. Binding of proteins is primarily due to half-ester sulfate groups, which are strongly anionic, being comparable with sulfuric acid. The action is considered to be electrostatic ion exchange. Treating the kappa-carrageenan with tannin blocks the sulfate groups to provide a treated carrageenan which is also usable as a specific adsorbent for proteins. Specifically, adsorption of glucoamylase on tannin-carrageenan beads occurred with an increased absorption capacity than other tannin-treated polysaccharides.
Agarose is a polysaccharide useful for binding of lysozyme and other proteins, and comprises (1,3)N-beta-D-galactopyranose-(1,4)N-3,6-anhydro-alpha-D-galactose. Chitosan is a polysaccharide essentially being a polymer of glucosamine subunits.